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Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system

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Abstract

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.

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Acknowledgments

The authors thank Dr. Alfred M. Handler (USDA/ARS, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida, USA) for kindly providing the pKhsp82-FLP Plasmid and Dr. Mark L. Siegald (Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA) for kindly providing the pMLS104 Plasmid. This work was supported by the Fundamental Research Funds for the Central Universities (XDJK2016C089), Project funded by China Post-doctoral Science Foundation (2015M580768), the China Agriculture Research System (CARS-22-ZJ0102).

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  1. State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Beibei, Chongqing, 400716, People’s Republic of China

    Dingpei Long, Weijian Lu, Zhanzhang Hao, Zhonghuai Xiang & Aichun Zhao

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  1. Dingpei Long
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  2. Weijian Lu
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  3. Zhanzhang Hao
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Correspondence to Aichun Zhao.

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Fig. S1

Structures of piggyBac-derived vectors and FLP recombinase or mRNA transient expression helper vectors (PDF 945 kb)

Fig. S2

Procedure for producing SSRS silkworms via sexual hybridization. (A) Strategy for producing SSRS silkworms via crossing heterozygous G1 TTS females with H-A males. One G1 heterozygous H-A male (♂) was backcrossed with three different G1 TTS females (♀) to produce three G2 broods (a, b, and c). The G2 eggs from each brood were treated with HCl solution to break the diapause. The hybrid TTS&H-A double-transgenic individuals were screened from each G2 brood and backcrossed with wild-type Dazao adults to produce G3 broods. These G3 broods were treated with HCl solution to break the diapause, and the fluorescence phenotypes of the larvae from these G3 broods were analyzed. (B) Strategy for production of SSRS silkworms by crossing heterozygous G1 TTS females with H–H males. One heterozygous G1 H–H male (♂) was backcrossed with three G1 TTS females (♀) to produce three G2 broods (d, e, and f). The G2 eggs from each brood were divided into two groups (numbered 1 and 2) and treated with HCl solution to break the diapause. Then, the three-day-old G2 eggs from group 2 of each brood were subjected to the HST three times per day at 6-h intervals for 5 days. After heat shock, the G2 eggs were maintained and reared at 25 °C. The eggs from group 1 of each G2 brood that were not subjected to the HST (no HST) were used as controls. The hybrid TTS&H–H double-transgenic individuals were screened from the d, e, and f broods of each group, and these G2 fertile adults were backcrossed with wild-type Dazao adults to produce G3 broods. These G3 broods were treated with HCl solution to break the diapause, and the fluorescence phenotypes of the larvae from these G3 broods of each group (G3 d, e, and f broods) were analyzed (PDF 1255 kb)

Fig. S3

Molecular confirmation of transgene constructs in the genomes of FLP recombinase expression helper strains. (A) Schematic maps of the transgene constructs in the genomes of H-A (top) and H–H individuals (bottom). The A3-MF/FLP-MR and Hsp-MF/FLP-MR primer pairs were used for PCR analysis of genomic DNAs from H-A and H–H individuals, respectively. (B) PCR analysis of genomic DNA using primer pairs specific for the H-A and H–H genomes. The expected 652-bp PCR products were observed for H-A-1–H-A-3 individuals using the A3-MF/FLP-MR primer pair. The expected 203-bp PCR products were observed for H–H-1 and H–H-2 individuals using the Hsp-MF/FLP-MR primer pair. Lane WT, the wild-type Dazao strain used as a control; lane M, Trans2 K Plus DNA Marker (PDF 579 kb)

Fig. S4

Expression of EGFP and DsRed genes in the larvae of the TTS and H-A-1 (or H–H-1) silkworms. Two-day-old first instar larvae of TTS (A, B) and H-A-1 (or H–H-1) silkworms (C, D) showing white light (A, C), GFP fluorescence (B), and RFP fluorescence (D) in the developing larval ocelli or epidermis. Arrowheads denote the positions of the GFP and RFP fluorescence in the larval ocelli; triangles denote GFP fluorescence in the larval epidermis. The different scale bars are located at the bottom left of the images (PDF 1933 kb)

Fig. S5

Strategy for FLP recombinase-mediated, site-specific excision of a target gene by sexual hybridization. (A) The mechanism of site-specific gene excision in the offspring of TTS&H-A-1 double-transgenic silkworms. A TTS adult was crossed with the recombinase-expressing helper strain H-A-1 adult to produce hybrid TTS&H-A-1 double-transgenic offspring. Recombination between two FRT sites of the TTS&H-A-1 germ cell genome, which was mediated by the A3 promoter driving FLP recombinase expression, resulted in deletion of the A3-EGFP expression cassette in the genome of SSRS individuals. (B) The mechanism of site-specific gene excision in the offspring of TTS&H–H-1 double-transgenic silkworms. A TTS adult was crossed with a recombinase-expressing helper strain H–H-1 adult to produce hybrid TTS&H–H-1 double-transgenic offspring. Recombination between two FRT sites of the TTS&H–H-1 germ cell genome, which was mediated by the hsp70 promoter driving FLP recombinase expression (HST), also resulted in the deletion of the A3-EGFP expression cassette in the genome of SSRS individuals (PDF 687 kb)

Fig. S6

Expression of EGFP gene in adult TTS and SSRS individuals. (A–D) show white light (A, C) and GFP fluorescent (B, D) images of the TTS adults. (E–H) show white light (E, G) and GFP fluorescent (F, H) images of the SSRS adults. Arrowheads denote the position of GFP fluorescence in the compound eye; triangles denote the position of GFP fluorescence in the wing epidermis. The white scale bar represents 1 mm (PDF 1410 kb)

Fig. S7

Sequencing of the FRT sites from TTS individuals and all of the SSRS individuals. Sequencing of the PCR products indicated that the structures of the two 48-bp FRT sites and restriction enzyme sites in the TTS genome (top), one 48-bp FRT site, and restriction enzyme sites in the SSRS genome (bottom) were as expected for a FLP recombinase-mediated, site-specific DNA excision event (PDF 795 kb)

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Long, D., Lu, W., Hao, Z. et al. Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system. Transgenic Res 25, 795–811 (2016). https://doi.org/10.1007/s11248-016-9970-4

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